1. Field of the Invention
Epidermal growth factor (EGF) is a polypeptide of 53 amino acids that has been characterized in both mice and humans. It is a potent mitogen for a variety of cells, such as fibroblasts, glia, epithelial, endothelial and epidermal cells, both cultured and in vivo. EGF is also a potent inhibitor of gastric acid secretion. EGF was first isolated from male mouse submaxillary glands, where it exists in inexplicably high levels.
In glandular homogenates, EGF is found as a 74,000 dalton complex of two molecules of EGF (Mr 6045) and two molecules of a binding protein (Mr 29,300), a kallikrein-like arginyl enteropeptidase The amino acid sequence of mouse submaxillary EGF has been determined and the synthesis of EGF and a larger 9000 dalton precursor with a carboxy terminal extension has been demonstrated in cultured submaxillary glands.
Human EGF, which appears to be similar if not identical to urogastrone, is also found in urine in larger forms of 28,000 and 30,000 Daltons that do not dissociate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Isolating both the DNA or RNA encoding for EGF and particularly a putative EGF precursor protein is extremely difficult for a number of reasons. Even where the peptide is abundant, the amount of messenger RNA is extremely small. Immunoprecipitation of in vitro translation products, even under strongly denaturing conditions fails to detect a precursor protein, possibly due to the huge size of the precursor and/or the masking of its antigenic determinants on the native peptide to which antibodies were made. Because of the physiological importance of EGF there is substantial interest in being able to obtain DNA sequences encoding for EGF and the EGF polypeptide precursor. In addition, since it is known that a number of hormones are generated by proteolytic processing from larger precursors the cDNA and derived amino acid sequence of the EGF precursor could reveal "cryptic", previously unknown polypeptide hormones and/or growth factors.
2. Description of the Prior Art
A human genomic cDNA library in bacteriophage .lambda. is described in Lawn et al., Cell (1978) 15:1157-1174. Savage et al., J. Biol. Chem. (1972) 247:7612-7621 report the amino acid sequence of mouse EGF. Sporn et al., Science (1983) 219:1329-1331 and Assoian et al., J. Biol. Chem. (1983) 258:7155-7160 describe transforming growth factors (TGF). See particularly Gray et al., Nature (1983) 303:722-725.